Rotaviruses (RVs), members of the Reoviridae family, have genomes consisting of eleven segments of double-stranded (ds) RNA. In the infectious RV particle, the genome is contained within a non-enveloped icosahedral capsid composed of three concentric protein layers. The innermost protein layer is a smooth, thin, pseudo T=1 assembly formed from 12 decamers of the core lattice protein VP2. Tethered to the underside of VP2 layer are complexes comprised of the viral RNA-dependent RNA polymerase (RdRP), VP1, and the RNA-capping enzyme, VP3. Together, VP1, VP2, VP3, and the dsRNA genome form the core of the virion. The core proteins function together to transcribe the segmented dsRNA genome, producing eleven capped plus-sense (+)RNAs. The viral RdRP uses the (+)RNAs as templates for the synthesis of the dsRNA genome. Although the RdRP alone can recognize viral (+)RNAs, the polymerase is only active when VP2 is present. The VP2-dependent activity of VP1 provides a means by which genome replication (dsRNA synthesis) can be linked with genome packaging (core assembly). Newly made (+)RNAs pass directly from the RdRP to VP3, an enzyme which introduces m7G caps at the 5'-end of the transcripts through associated guanylyltransferase and methyltransferase activities. Genome replication and core assembly take place in cytoplasmic inclusions bodies, termed viroplasms. Two viral nonstructural proteins, the octamer NSP2 and the dimer NSP5, direct the formation of viroplasms. The interactions of NSP2 and NSP5 with VP1, VP2, and VP3 coordinate genome replication and core assembly. The overriding goal of this project is to characterize the structure and function of the core proteins VP1, VP2, and VP3 and the viroplasm building-blocks NSP2 and NSP5. This includes defining the structural interfaces between the proteins and establishing how these interactions affect and regulate the activities of the proteins. Progress toward this goal is summarized below. (1) RNA interactions with the RV RdRP. The atomic structure of VP1 was determined by X-ray crystallography though a collaboration with Dr. Steve Harrison's group at Harvard, with details described in a publication appearing in 2008. Briefly, the results showed that the RV polymerase is a compact globular protein with three distinct domains: (i) an N-terminal protruding domain, (ii) a polymerase domain comprised of fingers, palm, and thumb subdomains, and (iii) a C-terminal bracelet domain. Together, the N- and C-terminal domains of VP1 sandwich most of the polymerase domain, creating a cage structure with the catalytic region located within a largely hollow center. Four tunnels connect the surface of VP1 to the catalytic center. These tunnels allow for (i) entry of nucleotides, (ii) entry of single-stranded template RNA, (iii) exit of the dsRNA product or the (-)RNA template, and (iv) exit of (+)RNA transcripts. Soaks of VP1 crystals with various RNA oligonucleotides have provided insight into the mechanism by which the RV polymerase binds and recognizes its (+)RNA templates. These analyses have revealed that the RdRP anchors the 3'consensus sequence (3'CS: 5'-UGUGACC-3') of (+)RNAs into the template entry tunnel and catalytic center via stacking interactions and an extensive network of hydrogen bonds. These interactions include specific contacts with the UGUG bases and nonspecific contacts with the sugar-phosphate backbone of the GACC portions of the 3'CS. Remarkably, these interactions place the 3'-terminal residue of the 3'CS past the site in the catalytic center required to support RNA initiation. In the presence of VP2, it is thought that conformational changes occur that bring the 3'-terminal residue back into proper register to support initiation. To better understand the importance of the interactions between the 3'CS and the RdRP for genome replication, we engineered mutant VP1 proteins and assayed their capacity to synthesize dsRNA in vitro. Our results showed that, individually, mutation of residues that interact specifically with RNA bases did not diminish replication levels. However, simultaneous mutation led to significantly lower levels of dsRNA product, presumably due to impaired recruitment of (+)RNA templates. In contrast, point mutation of nonspecific RNA contact residues in VP1 led to severely diminished replication, likely as a result of improper positioning of templates at the catalytic site. A noteworthy exception was a K419A mutation that enhanced the initiation capacity and product elongation rate of VP1. The specific chemistry of Lys419 and its position at a narrow region of the template entry tunnel appear to contribute to its capacity to moderate replication. Together, our findings suggest that distinct classes of VP1 residues interact with (+)RNA to mediate template recognition and dsRNA synthesis, yet function in concert to promote viral replication at appropriate times and with ideal kinetics. The findings of our analysis have implications for the structure and function of other RdRPs (e.g., HCV, FDHV), as they contain many of the same RNA-contact residues that are used by the RV RdRP to recruit template RNAs into the catalytic center of the enzyme. (2) VP2-dependent activation of the RV RdRP. Most human RV isolates can be classified into one of three groups (A, B, or C). Their segmented genome allows RVs to readily exchange genetic material during co-infections. This reassortment process occurs between viruses belonging to the same group, but not for viruses belonging to different groups. This restriction might reflect the failure of the viral RdRP to recognize and replicate viral RNAs of a different group. To address this question, we have carried out experiments aimed at contrasting the sequences, structures, and functions of RdRPs belonging to RV groups A, B, and C. We found that conserved amino acid residues are located within the hollow center of VP1 near the active site, whereas variable, group-specific residues are mostly surface exposed. By creating a three dimensional homology model of the group C RdRP, based on an atomic structure determined for the group A RdRP, we obtained evidence that these RV RdRPs have nearly identical tertiary folds and share similar mechanisms of recognizing RNA templates. Consistent with their structural analysis, we determined that recombinant group A and C RdRPs are capable of replicating one anothers RNA templates in vitro. However, the activity of both RdRPs is strictly dependent on the presence of their cognate VP2 core lattice protein. That is, the group A RdRP has activity in the presence of group A VP2, but not group C VP2, and vice versa. Thus, the reassortment restriction between rotavirus groups may reflect the inability of their replication proteins to function together in support of genome replication. In the past year, we undertook a study in which we assayed the VP1 and VP2 proteins of various strains of group A and C RVs to determine the limitations of functional compatibility for in vitro dsRNA synthesis. By engineering chimeric group A/C VP2 proteins capable of activating non-cognate VP1, we delineated core shell subdomains important for turning on polymerase function. Our results demonstrate that the amino termini of VP2, which interact to form an internal protruding hub underneath each fivefold axis (vis--vis, a VP2 decamer) of the viral core, play an important but non-specific role in VP1 activation. The VP2 residues that correlate with polymerase-activation specificity are located on the inner face of the principal (scaffold) domain, within the apical and/or central subdomains. These results indicate that function of the RV polymerase requires multiple interactions with the VP2 decamer, including with regions of both the hub and scaffold domain.